On the basis of sequence alignments, the
pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996)
Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)
Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant
lysine and
proline residues in Motif I of RluA and TruB,
pseudouridine synthases belonging to different families. Contrary to our expectations, the altered
enzymes display only very mild kinetic impairment. Substitution of the aligned
lysine and
proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate
proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and
Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of
pseudouridine synthase function to certain physiologically important eukaryotic
proteins that contain Motif I, including the human
protein dyskerin, alteration of which leads to the disease
dyskeratosis congenita.