Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues.
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.
Spedaliere
C J
CJ
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Hamilton
C S
CS
Mueller
E G
EG
D020816:Amino Acid Motifs / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011392:Proline; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase; D008239:Lysine